During early vertebrate development, lens is induced to form from the head surface ectoderm by the presumptive retinal neuroectoderm. When the evaginating optic vesicle contacts the surface ectoderm, the ectoderm thickens and forms a lens placode. In mammals, the lens placode invaginates and forms the lens vesicle. The posterior lens cells stop proliferation and differentiate into lens fiber cells. The anterior lens cells form a relatively undifferentiated, proliferatively active lens epithelium. Our long-term goal is to identify developmental steps and molecular events necessary for lens formation. The primary focus of this research is the role and the mechanism of action of the forkhead gene Foxe3. This research is a natural continuation of our previous studies investigating the role of this gene in lens formation. Since normal development and maintenance of lens cells is critical for vision, it is essential to define the developmental processes and gene networks that govern the development and survival of lens cells. Several key genes control of the formation, proliferation and differentiation of lens cells. One of them is the forkhead gene Foxe3. This gene encodes a transcription factor and mutations in this gene cause abnormal lens development in mouse and human. The absence of Foxe3 function leads to changes in the proliferation, differentiation and survival of lens cells. How the loss of Foxe3 leads to all of these changes remains largely unknown. Since Foxe3 is a transcription factor, its effects on the physiology and morphology of lens cells has to be mediated by other genes. With few exceptions, the downstream mediators of Foxe3 are not known. To better understand how Foxe3 regulates lens development and why mutations in this gene lead to such dramatically abnormal lens development, we will in Specific Aim 1 identify genes that belong to the Foxe3 lens regulatory network by comparing transcriptomes of wild type and Foxe3-deficient lenses using deep RNA-sequencing. In Specific Aim 2, we will identify direct target genes of Foxe3 by chromatin immunoprecipitation combined with sequencing. These cutting edge techniques in molecular and developmental biology will help us identify genes that mediate Foxe3 function during lens development. In Specific Aim 3, we will correct the molecular and phenotypic lens defects in mice with mutant or absent Foxe3 using in utero gene therapy. This research will lead to a better understanding of lens development, as this gene is regulating the early critical steps in lens formation. However, since mutations in this gene cause abnormal eye development in mouse and human, this research will result in knowledge that will allow a better diagnosis and treatment of diseases of the eye in which the components of the Foxe3 regulatory network are mutated. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify genes and developmental processes that are responsible for mammalian lens development. Identification of these genes and developmental processes will lead to the better understanding of eye diseases. As a result, new diagnostic procedures and treatments for eye diseases will be developed.